The focus of this application is the full characterization of the structural, functional, and immunological properties of the transmembrane proteins of HIV-1 and related viruses. We have recently found that the native structure of gp41 of HIV-1 is a tetramer, and have shown that some human monoclonal antibodies react preferentially with tetrameric gp41. In the present proposal the higher-order structure of HIV-2, SIV, and various recombinant and mutant HIV-1 env gene constructs will be determined, and the kinetics of oligomerization analyzed. Tetrameric gp41 complexes will be purified, and used to develop a gp120-gp41 reconstitution assay. The reconstituted complexes will be fully characterized, and their antigenicities evaluated. They will also be used in photo-affinity labeling experiments, to detect and characterize potential cell-surface receptors other than CD4, which may play a role in the fusion reaction. The topography of gp41 will be determined by a combination of biochemical and immunological methods, and domains accessible on the surface of virions and infected cells will be identified. Similar experiments will be performed for HIV-2 and SIV. The complete secondary structure and post-translational modifications of gp41 will be analyzed, including disulfide bond organization, sites of glycosylation (both N- and O-linked), and lipid esterification. Structure-function analyses of these features will be performed by site-specific mutagenesis; this approach will also be used to analyze the role of the hydrophobic N-terminal sequence, and the C-terminal domain of gp41. The effects of these mutations on synthesis, transport, processing, tetramerization, and immunogenicity of the HIV env proteins, and on the infectivity, and cytotoxicity of the resulting viruses, will be determined. The cytotoxicity and possible virus-inhibitory effects of the N-terminal sequence of gp41 will be further defined, using synthetic peptides, and peptide analogues. Possible immunosuppressive activities of native gp41 and gp41 peptides will be determined; if we find significant activity, the structural requirements for this effect will be identified, and site-specific mutagenesis will be used to generate mutant gp41 proteins which cause negligible or reduced immunosuppression. Additional human nd rat monoclonal antibodies against the HIV env proteins will be characterized by epitope mapping, and tested for antiviral activities. Hybrid antibodies and antibody heteroconjugates will be prepared and characterized for enhanced affinity, neutralization, and cytotoxic effects. The efficacy of hybrid antibodies as agents for passive immunotherapy will be tested in tissue culture, and the effectiveness of reconstituted native env structures as vaccines will be determined in rodents. If results are promising, subsequent studies with both approaches will be performed in non-human primates, including chimpanzees, in collaboration with the Laboratory of Experimental Medicine and Surgery in Primates (LEMSIP).